The present invention relates to the field of somatic embryo production, particularly to methods for the regeneration of Acacia mangium through somatic embryogenesis. More specifically, the present invention relates to a method for regeneration of plants of Acacia mangium by culturing explants of immature zygotic embryos on callus induction medium to grow embryogenic tissue. Culturing of the embryogenic tissue is continued on somatic embryo maturation medium and germination medium. The germinated embryos are further converted to acclimatized plants for field planting. The method is well suited for producing clonal planting stock useful for reforestation.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
Forests are very important to the world economy and for maintaining and preserving our ecosystem. Forest trees have a wide range of commercial uses (timber for construction, raw material for paper and pulp production, and as an energy source). The global demand for wood products (mostly for paper and pulp and firewood in developing countries) has been increasing year by year when the natural forests are in short supply. Re-forestation is the solution to meeting such increasing demand. Usually, the fast-growing, widely adapted tree species are chosen for re-forestation. Most tree-improvement programs are based on the management of genetic resources, including the selection of superior clones from existing forests, the conservation of genetic variability, partially controlled propagation and classical breeding for desired traits. In spite of the fact that it usually takes several generations to breed, this traditional breeding has been successful in getting elite trees with fast and uniform growth. However, many other traits such as disease and insect resistance, different lignin composition and content are difficult to acquire mainly due to high heterozygosity in tree species and big segregation population. Moreover, the gene(s) conferring certain phenotypes like disease resistance may not be in the gene pool at all. On the other hand, molecular breeding based on genetic transformation of tree species offers the possibility to introduce a particular phenotype without affecting the genetic background of a cultivar. Genetic transformation in Populus species and Eucalyptus species enabled some success in modification of lignin content (Tzfira et al., 1998; Robinson, 1999). The precondition of molecular breeding of forest tree species is the availability of a reliable and reproducible genetic transformation method, which in turn relies on a system of regeneration of one whole plant from a single cell.
Genus Acacia comprises about 1200 tropical and subtropical tree species. It belongs to the family Mimosaceae. Acacia mangium is a multipurpose, fast growing and nitrogen fixing elite tropical legume tree. A. mangium has been increasingly used for plantation, reforestation and soil rehabilitation in degraded soil. Many A. mangium plantations have been established in acidic soil or abandoned land. Yielding high quality fibre, A. mangium has been increasingly planted in many regions of South East Asia like Malaysia (Tsai, 1988) and Indonesia as raw material for pulp industry. Many paper and pulp mills in Indonesia have been increasingly relying on plantation as the source of wood and A. mangium is the preferred choice. Asia Paper and Pulp group has two affiliate companies with total concession of 540,000 hectares. By 1996, one company had planted 123,000 hectares of A. mangium, about 90% of its plantation, which represented 180 million seedlings. It was estimated that by 2004, Asia Paper and Pulp group will virtually source all its wood from plantation, mainly A. mangium plantation (Bayliss, 1998a, 1998b). However, the flowers of A. mangium show weak protogyny and variable levels of andromonoecy. It possesses the characteristics of self-pollination, cross-intraspecific pollination or interspecific pollination with A. auriculiformis in nature (Sedgley et al., 1992; Sornsathapornkul and Owens, 1999). These characteristics of reproduction are disadvantageous to commercial propagation and plantation through seeds.
Regeneration is often used in woody tree propagation (Kozlowski and Pallardy, 1997). In Acacia, a few species were reported the success of regeneration, such as Acacia catechure regeneration via somatic embryogenesis (Rout et al., 1995) and A. auriculiformis regeneration through organogenesis (Rao and Prasad, 1991). A. mangium shoot propagation was reported (Bhaskan and Subbash, 1996; Ahmad, 1991; Galiana et al., 1991a, 1991b;) and Toshihiro (1999) also reported the isolation of protoplast from sterile A. mangium seedlings. Our PCT patent application No. PCT/SG00/00010 describes A. mangium regeneration through organogenesis. However, regeneration of A. mangium through somatic embryogenesis has not been described.
Somatic embryos are clonal in origin and thus multiplication using somatic embryos can have the potential for exceedingly high rates of vegetative increase and is therefore of considerable commercial interest. Regeneration via somatic embryogenesis is an attractive option for plant tissue culture. Somatic embryos reportedly provide more stable regenerants than shoots. Another advantage of regeneration systems using somatic embryos is their apparent single cell origin. This means that it is unlikely that regenerants are of chimerical origin, since, if a regenerant originates from a cluster of cells rather than a single sell, the plant tissues may be chimerical or unstable and produce off-types. The availability of somatic embryogenesis protocols for potato, or other crop species recalcitrant to somatic embryogenesis, will permit these crops to take advantage of any new artificial seed technology advances.
Somatic embryos are suitable for transformation via Agrobacterium tuniefaciens (Mathews et al., 1992), microinjection (Neuhaus et al., 1987) and particle bombardment (Wilde et al., 1992). In addition, somatic embryos can be cryopreserved using liquid nitrogen without loss of viability. Cryopreservation is an efficient means of maintaining germplasm and enables plant material to be transported over large distances. Furthermore, somatic embryos are suitable for the development of artificial seed technology (U.S. Pat. No. 5,572,827; Bajaj, 1995a).
Thus, it is an object of the present invention to provide a method for regeneration of A. mangium through somatic embryogenesis.
The present invention relates to the field of somatic embryo production, particularly to methods for the regeneration of Acacia mangium through somatic embryogenesis. More specifically, the somatic embryogenesis process for A. mangium of the present invention can be divided into four general steps: (1) embryogenic callus induction, (2) embryo maturation, (3) embryo germination, and (4) conversion into acclimatized plants, i.e., regenerated plants or regenerants. The acclimatized plants are then transferred to the field. It is preferred to use a two step embryo maturation.
The present invention also relates to a process for preparing somatic embryos for A. mangium. This process generally comprises the first two previously described steps. The somatic embryos can be used as a source material for preparing transgenic plants, can be cryopreserved and can be developed into artificial seed.
According to one embodiment of the present invention, somatic embryogenesis of Acacia mangium is achieved from callus induced from immature embryo axes on MS basal medium supplemented with the combinations of thidiazuron (IDZ) and indole-3-acetic acid (IAA) together with ascorbic acid, asparagine, casein enzymatic hydrolysate, glutamine, proline, sucrose and phytagel. Through two steps of maturation procedure by culturing somatic embryos first on xc2xd MS basal medium containing sucrose, phytagel and gibberellic acid (GA3) and then on xc2xd basal medium containing sucrose and phytagel, 42.33% of globular embryos developed into torpedo and cotyledon embryos. 11% of mature somatic embryos germinated into seedlings that were transferred to pot soil consisting of a mixture of peat soil and sand.